The detection of a clade 2.3.4.4b influenza A(H5N1) virus bearing an HA-E190D substitution in an infected human raises concern about mammalian adaptation. We evaluated the impact of the naturally occurring HA-E190D substitution in the A/British Columbia/PHL-2032/2024 (BC/24) virus on receptor binding specificity and viral fitness in vitro and in vivo. Recombinant BC/24 HA-E190D protein retained α2,3-linked sialic acid binding specificity but with reduced binding affinity. In cell culture, BC/24 viruses dominated by HA-190D were frequently outcompeted by the minor HA-190E variant in the inoculum, whereas BC/24 HA-190E viruses maintained dominance in the presence of residual HA-190D in the inoculum. In ferrets, BC/24 HA-190D viruses were similarly outcompeted by HA-190E; in one out of six ferrets where HA-190D dominance persisted, the virus was attenuated and failed to spread systemically. In contrast, BC/24 HA-190E viruses maintained dominance and disseminated systemically. These results indicate that the BC/24 variant bearing HA-E190D substitution did not acquire human-like receptor binding specificity and was less fit in mammalian hosts, making it unlikely to enhance public health risk without additional compensatory mutations.